Peptide derivatives for treatment, prevention or alleviation of a condition associated with bone loss or low bone density or to inhibit osteoclast differentiation and stimulation

ABSTRACT

Compounds of formula R 1 -A-NH—CH(R 2 )—CO—R 3  are presented. The compounds can be used to treat or alleviate conditions associated with bone loss or low bone density, or to inhibit osteoclast differentiation and stimulation, bone resorption and/or loosening of a prosthetic device. The compounds can be administered as a pharmaceutical composition or in a prosthetic device.

FIELD OF INVENTION

The invention relates to novel compounds which may be for treatment,prevention or alleviation of a condition associated with bone loss orlow bone density or to inhibit osteoclast differentiation andstimulation, bone resorption, or loosening of a prosthetic device.

BACKGROUND OF INVENTION

There is an increasing demand for the development of compounds havingimproved properties and which can be used against several differentdiseases, such as treatment of an infection or disease caused by amicroorganism as well as for the stimulation or inhibition of theproliferation of eukaryotic cells as well as for other purposes.

One class of compounds, namely peptide derivatives based upon theinhibitory centre of human cystatin C, and the antibacterial propertiesthereof have been disclosed in Kasprzykowski et al., APMIS 2000, 108,473-481.

WO 06/052201 discloses the use of peptide derivatives for themanufacture of a medicament for the treatment of microbial infections.

WO 07/129952 discloses novel peptide derivatives and the use thereof asantimicrobial agent for treatment of wounds.

Hitherto no suggestions have been given for use of the mentioned peptidederivatives other than for antimicrobial and antibacterial purposes.

Osteoclasts constitute one type of eukaryotic cells. Bone resorption isa specific function of osteoclasts, which are multinucleated,specialized bone cells formed by the fusion of mononuclear progenitorsoriginating from the haemopoietic compartment, more precisely from thegranulocyte-macrophage colony-forming unit (GM-CFU). The osteoclast isthe principal cell type for resorption of bone. Osteoclasts togetherwith the bone-forming cells, the osteoblasts, dictate bone mass, boneshape and bone structure. Bone must undergo continuous resorption andrenewal, a process collectively known as remodelling. During adult lifebone remodelling is crucial to eliminate and replace structurallydamaged or aged bone with structurally new healthy bone. To maintain theproper bone mass, resorption and formation are kept in perfectequilibrium. The balanced bone remodelling is disturbed in certainpathological conditions either to systemic excess or decrease ofendocrine factors or the presence of local pathological conditions inthe skeleton. In such diseases, the equilibrium between bone resorptionand formation becomes altered, often in favour of resorption, resultingin a reduction in bone mass, deterioration of bone architecture,decreased resistance to stress, bone fragility, susceptibility tofractures or to disabilities in joints or teeth. Hence, increasedactivity and/or numbers of osteoclasts, relative to the activity and/ornumbers of osteoblasts, may lead to a pathological loss of bone.

For conditions in which osteoclasts resorb bone at abnormally highlevels as in osteoporosis, rheumatoid arthritis, periodontal disease,metastatic tumours, loss of joint prosthesis or tooth implant loss, themost reasonable therapeutic target would be the osteoclast. Decreasingthe number of osteoclasts and/or the resorption activity of theosteoclasts, should restore the equilibrium between bone resorption andformation.

SUMMARY OF THE INVENTION

The object of the present invention is a number of invented compoundswhich prevents osteoclast formation and/or inhibits proteases, such ascystein proteases, which may be used for the manufacture of a medicamentfor treatment, prevention or alleviation of a condition associated withbone loss or low bone density or to inhibit osteoclast differentiationand stimulation, bone resorption, or loosening of a prosthetic device.

The invention relates in one aspect to a compound of the general formula(I)R¹-A-NH—CH(R²)—CO—R³

-   wherein-   R¹ is Ar—X—, where X is (CH₂)_(m)—O—CO; (CH₂)_(m)—SO₂—CH═CH—CO;    CH═CH—CO; CO—CH═CH—CO; CO-oxirane-CO; or-   R¹ is Hal-(CH₂)_(m)—CO, where Hal is F, Cl, Br or I; or-   R¹ is CH₃—O—CO-oxirane-CO; or-   R¹ is NH₂—C(NH)—NH—(CH₂)_(m)—CO; or-   R¹ is an acyl residue containing coumarin moiety, optionally    hydroxylated;-   A is a bond or is one or more amino acid such as Arg, Val or Leu;-   R² is isopropyl, sec-butyl or isobutyl;-   R³ is Pro or Arg-B, where B is NH—NH—CO-Ile-Val-O—CH₃; or-   R³ is NH—(CH₂)_(m)—NH—C(NH)—NH₂; or-   R³ is NH—CH(R⁴)—(CH₂)_(m)—NH-D, where R⁴ is C₁-C₆-alkyl, such as    isopropyl, and D is CO—CH═CH—Ar or CO—CH═CH—SO₂—(CH₂)_(m)—Ar, or    Cum-Phe- sequence, where Cum is an acyl residue containing coumarin    moiety, optionally hydroxylated;-   Ar is aryl or heteroaryl, such as phenyl or pyridyl;-   m is an integer of 0 to 5;-   or-   wherein-   R¹ is Ar—X—, where X is (CH₂)_(m)—O—CO; (CH₂)_(m)—SO₂—CH═CH—CO;    CH═CH—CO; CO-oxirane-CO; CO—CH═CH—CO; CH₂—CH═CH—CO; (CH₂)_(m)—CO or-   R¹ is CH₃—O—CO-oxirane-CO; or-   R¹ is NH₂—C(NH)—NH—(CH₂)_(m)—CO; or-   R¹ is an acyl residue containing coumarin moiety, optionally    hydroxylated;-   A is a bond or is one or more amino acid such as Arg, Val or Leu;-   R² is isopropyl or isobutyl;-   R³ is NH—(CH₂)_(m)—NH—C(NH)—NH₂; or-   R³ is Phe-NH₂; or-   R³ is NH—CH(R⁴)—(CH₂)_(m)—NH-D, where R⁴ is C₁-C₆-alkyl, such as    isopropyl, and D is CO—CH═CH—Ar or CO—CH═CH—SO₂—(CH₂)_(m)—Ar, or    Cum-Phe- sequence, where Cum is an acyl residue containing coumarin    moiety, optionally hydroxylated;-   Ar is aryl or heteroaryl, such as phenyl or pyridyl;-   m is an integer of 0 to 5;-   as a single stereoisomer or a mixture of different stereoisomers;-   or a pharmaceutically acceptable salt thereof.

By the new invented compounds it is for the first time possible totreat, prevent or alleviate conditions associated with bone loss or lowbone density or to inhibit osteoclast differentiation and stimulation,bone resorption, or loosening of a prosthetic device.

Further advantages and objects with the present invention will bedescribed in more detail, inter alia with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows osteoclast formation.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention, the followingdefinitions apply:

The term “pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution, saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

In the present application an arrow “←” found when defining a sequenceindicates the direction of the amide bond of the subsequent group.

In the present application the expression “Valψ(CH₂NH)” means that thepeptide (amide) bond is reduced.

The Compound

The invention relates to the unexpected finding that the compounds ofthe invention having the general formula (I)R¹-A-NH—CH(R²)—CO—R³

-   wherein-   R¹ is Ar—X—, where X is (CH₂)_(m)—O—CO; (CH₂)_(m)—SO₂—CH═CH—CO;    CH═CH—CO; CO—CH═CH—CO; CO-oxirane-CO; or-   R¹ is Hal-(CH₂)_(m)—CO, where Hal is F, Cl, Br or I; or-   R¹ is CH₃—O—CO-oxirane-CO; or-   R¹ is NH₂—C(NH)—NH—(CH₂)_(m)—CO; or-   R¹ is an acyl residue containing coumarin moiety, optionally    hydroxylated;-   A is a bond or is one or more amino acid such as Arg, Val or Leu;-   R² is isopropyl, sec-butyl or isobutyl;-   R³ is Pro or Arg-B, where B is NH—NH—CO-Ile-Val-O—CH₃; or-   R³ is NH—(CH₂)_(m)—NH—C(NH)—NH₂; or-   R³ is NH—CH(R⁴)—(CH₂)_(m)—NH-D, where R⁴ is C₁-C₆-alkyl, such as    isopropyl, and D is CO—CH═CH—Ar, CO—CH═CH—SO₂—(CH₂)_(m)—Ar, or    Cum-Phe- sequence, where-   Cum is an acyl residue containing coumarin moiety, optionally    hydroxylated;-   Ar is aryl or heteroaryl, such as phenyl or pyridyl;-   m is an integer of 0 to 5;-   or-   wherein-   R¹ is Ar—X—, where X is (CH₂)_(m)—O—CO; (CH₂)_(m)—SO₂—CH═CH—CO;    CH═CH—CO; CO-oxirane-CO; CO—CH═CH—CO, CH₂—CH═CH—CO; (CH₂)_(m)—CO or-   R¹ is CH₃—O—CO-oxirane-CO; or-   R¹ is NH₂—C(NH)—NH—(CH₂)_(m)—CO; or-   R¹ is an acyl residue containing coumarin moiety, optionally    hydroxylated;-   A is a bond or is one or more amino acid such as Arg, Val or Leu;-   R² is isopropyl or isobutyl;-   R³ is NH—(CH₂)_(m)—NH—C(NH)—NH₂; or-   R³ is Phe-NH₂; or-   R³ is NH—CH(R⁴)—(CH₂)_(m)—NH-D, where R⁴ is C₁-C₆-alkyl, such as    isopropyl, and D is CO—CH═CH—Ar, CO—CH═CH—SO₂—(CH₂)_(m)—Ar, or    Cum-Phe- sequence, where Cum is an acyl residue containing coumarin    moiety, optionally hydroxylated;-   Ar is aryl or heteroaryl, such as phenyl or pyridyl;-   m is an integer of 0 to 5;-   as a single stereoisomer or a mixture of different stereoisomers;-   or a pharmaceutically acceptable salt thereof, results in inhibition    of osteoclast differentiation, formation, or function, leads to less    bone resorption, As such, these compounds may be used to treat a    subject having a condition characterized by bone loss. These    compounds may be especially well suited for treatment, prevention or    alleviation of a condition associated with systemic bone loss or low    bone density or to inhibit osteoclast differentiation and    stimulation, bone resorption, or loosening of a prosthetic device,    such as a bone disease associated with primary or secondary    osteoporosis, juvenile osteoporosis, osteogenesis imperfecta,    hypercalcaemia, hyperparathyroidism, osteomalacia,    osteohalisteresis, osteolytic bone disease, osteonecrosis and    Paget's disease of bone or a condition being a secondary    osteoporosis such as bone loss due to rheumatoid arthritis,    inflammatory arthritis, osteomyelitis, bone loss due to an eating    disorder, metastatic bone diseases, periodontal bone loss, bone loss    due to cancer and age-related loss of bone or bone resorption    induced by pharmaceuticals or implantates. Examples of implantates    include prosthetic joint implantation as well as dental and other    implantations. Other conditions where facilitation of bone repair or    replacement is desired such as bone fractures, bone defects, plastic    surgery. It was determined that the invented compounds inhibit    osteoclast formation. These results suggest that the invented    compounds may be useful in the treatment of all the    diseases/disorder or conditions mentioned above.

FIG. 1 describes that steoclasts are giant cells with several nuclei andthey are the only cells in nature which can resorb bone (breakdown ofbone) and they do so by adhering to bone tissue and creating aresorption lacunae. The bone tissue is resorb in the lacuane by a twostep process in which the osteoclasts dissolve mineral crystals bycreating an acid pH in the resorption lacunae and degrade the boneprotein matrix by releasing proteolytic enzymes, including cysteineproteinases like cathepsin K. These giant cells are formed frommononuclear precursor cells from bone marrow. It is required that suchprecursor cells are stimulated by the cytokines M-CSF (macrophagecolony-stimulating factor) and RANKL (receptor activator of nuclearfactor kappa B ligand). M-CSF is needed for the precursor cells toproliferate and RANKL for that they can differentiate (becomespecialized) to cells which eventually fuse to the inactive osteoclastswhich then can adhere to bone and become active. The compounds of theinvention do not affect the proliferative step but inhibits thedifferentiation pathway.

In specific embodiments R¹ is Ar—(CH₂)_(m)—O—CO orNH₂—CH(NH)—NH—(CH₂)_(m)—CO and/or R² is isobutyl and/or R³ isNH—(CH₂)_(m)—NH—CH(NH)—NH₂. In one specific embodiment R¹ is(7-hydroxycoumarin-4-yl)acetyl residue.

The compound according to the present invention may be selected from anyof the following:

-   as a single stereoisomer or a mixture of different stereoisomers;-   or a pharmaceutically acceptable salt thereof.

In one embodiment the compound is selected from any of the followingcompounds:

-   Z-RLR, M-1; A11, A12, A25, A33, A42, A47, A50, A54, A107, A127 and    A128 as a single stereoisomer or a mixture of different    stereoisomers or a pharmaceutically acceptable salt thereof. Said    compounds are as defined above.

In another embodiment the compound is selected from the following:

-   Z-RLR, M-1, A11, A-47, A-54, A-107, A-127 and A-128 as a single    stereoisomer or a mixture of different stereoisomers; or a    pharmaceutically acceptable salt thereof.

In a further embodiment the compound is selected from the followingcompounds:

-   A-20, A-25, A-26, A-30, A-33, A-47, A-49, A-50, A-54, A-59, A-107,    A-116, A-118, A-119, A-120, A-123, A-127 and A-128 as a single    stereoisomer or a mixture of different stereoisomers; or a    pharmaceutically acceptable salt thereof.

In another embodiment the compound is selected from any of the followingcompounds:

-   A-20, A-25, A-26, A-30, A-33, A-47, A-49, A-50, A-54, A-59 as a    single stereoisomer or a mixture of different stereoisomers; or a    pharmaceutically acceptable salt thereof.

In yet another embodiment the compound is selected from any of thefollowing compounds:

-   A-59, A-107, A-116, A-118, A-119, A-120, A-123, A-127 and A-128 as a    single stereoisomer or a mixture of different stereoisomers; or a    pharmaceutically acceptable salt thereof.

In yet another embodiment the compound is the compound denoted A59 asdefined above, as a single stereoisomer or a mixture of differentstereoisomers; or a pharmaceutically acceptable salt thereof.

The compounds according to the present invention are potential cysteineproteases inhibitors and/or possesses antimicrobial properties, forexample compounds denoted A-25 A-33, A-54, A-59 and A-107 and A-20

The invention also relates to the use of any of the above mentionedcompounds or a compound selected from any of the following:

-   as a single stereoisomer or a mixture of different stereoisomers; or    a pharmaceutically acceptable salt thereof, for the manufacture of a    medicament for treatment, prevention or alleviation of a condition    associated with bone loss or low bone density or to inhibit    osteoclast differentiation and stimulation, bone resorption, or    loosening of a prosthetic device, wherein the condition is a bone    disease selected from osteoporosis, juvenile osteoporosis,    osteogenesis imperfecta, hypercalcaemia, hyperparathyroidism,    osteomalacia, osteohalisteresis, osteolytic bone disease,    osteonecrosis and Paget's disease of bone or a condition being a    secondary osteoporosis such as bone loss due to rheumatoid    arthritis, inflammatory arthritis, osteomyelitis, bone loss due to    an eating disorder, metastatic bone diseases, periodontal bone loss,    bone loss due to cancer and age-related loss of bone or bone    resorption induced by pharmaceuticals or implantates.    Pharmaceutical Compositions

Candidates for therapy with the compounds/agents identified by themethods described herein are patients either suffering from bone loss,bone resorption or patients who have a medical prosthesis implanted orwho contemplate receiving an implant medical prosthetic device. Thepharmaceutical formulation may be administrated when a candidate sufferfrom a disease or disorder as well as be given prophylactic.

The invention provides methods of treatment featuring administering to asubject an effective amount of a compound/agent of the invention. Thecompound is preferably substantially purified (e.g., substantially freefrom substances that limit its effect or produce undesiredside-effects). The subject is preferably an animal, including but notlimited to animals such as monkeys, cows, pigs, horses, chickens, cats,dogs, etc., and is preferably a mammal, and most preferably human. Inone specific embodiment, a non-human mammal is the subject. In anotherspecific embodiment, a human mammal is the subject. Accordingly, thecompound/agents synthesized by the methods described herein may beformulated as pharmaceutical compositions to be used for prophylaxis ortherapeutic use to treat these patients.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, or microcapsules. Methods of introduction can be enteralor parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, topical and oral routes. The compounds may be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. In a specific embodiment, it may be desirable toadminister the pharmaceutical compositions of the invention locally tothe area in need of treatment.

Such compositions comprise a therapeutically effective amount of anagent, and a pharmaceutically acceptable carrier. In a particularembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilisingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

In another embodiment, the compound can be delivered in a vesicle, inparticular a liposome (Langer (1990) Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327)

In yet another embodiment, the compound can be delivered in a controlledor sustained release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(See, Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger et al., (1983) Macromol. Sci.Rev. Macromol. Chem. 23:61; Levy et al. (1985) Science 228:190; Duringet al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg.71:105). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the subject bone orprosthesis, thus requiring only a fraction of the systemic dose (see,e.g., Goodson, in Medical Applications of Controlled Release (1984)supra, vol. 2, pp. 115-138). Other suitable controlled release systemsare discussed in the review by Langer (1990) Science 249:1527-1533.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of thecompounds described herein as an active ingredient.

The present compounds or agents that are described herein can be used asthe sole active agents, or can be used in combination with one or moreother active ingredients. These agents are known in the art, and can beselected from anti-inflammatory compounds, bisphosphonates, solubleRANK, anti-RANKL, OPG and bone morphogenetic proteins, for instance.

When contemplating combination therapy with the compounds defined hereinand one or more of the above-noted agents, it is important to assessclinical safety by methods known to those skilled in the art.Appropriate dose titration may be necessary when certain groups ofcompounds are contemplated for use together.

The compounds or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Preferably, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treating it with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In thepreferred embodiment, the matrix is biodegradable over a time period ofless than a year, more preferably less than six months, most preferablyover two to ten weeks. The polymer composition, as well as method ofmanufacture, can be used to determine the rate of degradation. Forexample, mixing increasing amounts of polylactic acid with polyglycolicacid decreases the degradation time. Meshes of polyglycolic acid thatcan be used can be obtained commercially, for instance, from surgicalsupply companies (e.g., Ethicon, N.J.). A hydrogel is defined as asubstance formed when an organic polymer (natural or synthetic) iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure which entraps water moleculesto form a gel. In general, these polymers are at least partially solublein aqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, which have charged side groups, or a monovalent ionicsalt thereof.

For use in treating animal subjects, the compositions of the inventioncan be formulated as pharmaceutical or veterinary compositions.Depending on the subject to be treated, the mode of administration, andthe type of treatment desired, e.g., prevention, prophylaxis, therapy;the compositions are formulated in ways consonant with these parameters.A summary of such techniques is found in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co., Easton, Pa.

The compositions of the present invention may be administeredparenterally, orally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Formulations may be prepared in a manner suitable forsystemic administration or for topical or local administration. Systemicformulations include, but are not limited to those designed forinjection (e.g., intramuscular, intravenous or subcutaneous injection)or may be prepared for transdermal, transmucosal, nasal, or oraladministration. Such compositions may be prepared as injectables, eitheras liquid solutions or suspensions, however, solid forms suitable forsolution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

The compounds defined above can be formulated into the therapeuticcomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the compounds) and which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formedfrom the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and the like. For oraladministration, the compositions can be administered also in liposomalcompositions or as microemulsions. Suitable forms include syrups,capsules, tablets, as is understood in the art.

The compositions of the present invention may also be administeredlocally to sites in subjects, both human and other vertebrates, such asdomestic animals, rodents and livestock, using a variety of techniquesknown to those skilled in the art. For example, these may includesprays, lotions, gels or other vehicles such as alcohols, polyglycols,esters, oils and silicones.

The administration of the compositions of the present invention may bepharmacokinetically and pharmacodynamically controlled by calibratingvarious parameters of administration, including the frequency, dosage,duration mode and route of administration. Variations in the dosage,duration and mode of administration may also be manipulated to producethe activity required.

The therapeutic compound compositions defined herein are conventionallyadministered in the form of a unit dose, for instance intravenously, asby injection of a unit dose, for example. The term “unit dose” when usedin reference to a therapeutic composition of the present inventionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the agentselected for treating the subject, the dosage formulation, and in atherapeutically effective amount. The desired effect refers to theeffect of the agent on reducing or inhibiting osteoclast differentiationand activity by reducing or inhibiting bone resorption. Moreover, thequantity of the compound to be administered depends on the subject to betreated as well as the extent or severity of bone resorption. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.However, suitable dosages to achieve the desired therapeutic effect invivo may range from about 0.1 mg/kg body weight per day to about 200mg/kg body weight per day, or from about 1.0 mg/kg body weight per dayto about 100 mg/kg body weight per day, preferably about 25 mg/kg bodyweight per day to about 50 mg/kg body weight per day. The preferred dosewill depend on the route of administration. However, dosage levels arehighly dependent on the nature of the disease or situation, thecondition of the subject, the judgment of the practitioner, and thefrequency and mode of administration. If the oral route is employed, theabsorption of the substance will be a factor effecting bioavailability.A low absorption will have the effect that in the gastro-intestinaltract higher concentrations, and thus higher dosages, will be necessary.Suitable regimes for initial administration and further administrationare also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain desired concentrations, e.g. in theblood, are contemplated. The composition may be administered as a singledose multiple doses or over an established period of time in aninfusion.

It will be understood that the appropriate dosage of the substanceshould suitably be assessed by performing animal model tests, where theeffective dose level (e.g., ED.sub.50) and the toxic dose level (e.g.TD.sub.50) as well as the lethal dose level (e.g. LD.sub.50 orLD.sub.10) are established in suitable and acceptable animal models.Further, if a substance has proven efficient in such animal tests,controlled clinical trials should be performed.

The compounds or compositions of the present invention may be modifiedor formulated for administration at the site of pathology. Suchmodification may include, for instance, formulation which facilitate orprolong the half-life of the compound or composition, particularly inthe environment. Additionally, such modification may include theformulation of a compound or composition to include a targeting proteinor sequence which facilitates or enhances the uptake of thecompound/composition to bone or bone precursor cells. In a particularembodiment, such modification results in the preferential targeting ofthe compound to bone or bone precursor cells versus other locations orcells. In one embodiment, a tetracycline, tetracycline family orbisphosphonate may be utilized to target the compound or composition ofthe present invention to bone or bone cells, including osteoclasts andosteoclast precursors. Novel heterocycles as bone targeting compoundsare disclosed in U.S. Patent Publication No. 2002/0103161 A.sub.1, whichis incorporated herein by reference in its entirety.

Pharmaceutically acceptable carriers useful in these pharmaceuticalcompositions include, e.g., ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Sterile injectable forms of the compositions may be aqueous oroleaginous suspensions. The suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered once a day or on an “as needed” basis.

The pharmaceutical compositions may be orally administered in any orallyacceptable dosage form including, capsules, tablets, aqueous suspensionsor solutions. In the case of tablets for oral use, carriers commonlyused include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavouring or colouring agents may also beadded.

Alternatively, the pharmaceutical compositions may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically. Topical application can be effected in a rectalsuppository formulation (see above) or in a suitable enema formulation.Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention includes, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilisingor dispersing agents.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects (a) approval by the agency ofmanufacture, use or sale for human administration, (b) directions foruse, or both.

The invention also provides prosthetic devices having one or more of thecompounds defined herein thereon or therein. The compound or compoundsmay be present in a composition applied to one or more surfaces of theprosthetic device or be present within the prosthetic device. That is,the compound or compounds, may be present within the very matrix of theprosthetic device such as for instance within the cement, e.g.methylmethacrylate cement that forms the prosthetic device.

Following examples are intended to illustrate, but not to limit, theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES

The following abbreviations are used in the examples: THF,tetrahydrofuran; HOBt, hydroxybenzotriazole; DCC,dicyclohexylcarbodiimide; DCU, dicyclohexylurea; TFA, trifluoroaceticacid; DMF, dimethylformamide; DIPEA, N,N-diisopropylethylamine;TBTU,O-(benzotriazol-1yl)-NNN′N′-tetramethyluronium tetrafluoroborate;DCM, dichlormethane, MeOH, methanol; DMSO, dimethylsulfoxide

Example 1 Synthesis of Coumarinacetyl-Containing Derivative A-1284-(tert-butyloxycarbonylamino)butylamine hydrochloride

The 4-(tert-butyloxycarbonylamino)butylamine hydrochloride was obtainedfrom 4-aminobutan-1-ol in accordance with procedures described inliterature (Mattingly, P. G., Synthesis, 1990, 1990, 366-368). m.p.156-158° C.

4-(benzyloxycarbonyl-L-leucylamino)-N-(tert-butoxycarbonyl)butylamine

To a vigorously stirred solution of4-(tert-butyloxycarbonylamino)-butylamine hydrochloride (0.140 g, 0.624mmol), Z-L-leucine (0.165 g, 0.624 mmol), HOBt (0.084 g, 0.624 mmol),triethylamine (174 μl, 1.25 mmol) in THF (25 ml) cooled on ice bath, DCC(0.141 g, 0.686 mmol) was added in small portions, during 0.5 h. Themixture was stirred on ice bath for additional 1 h, next left at roomtemperature overnight. The precipitated DCU was filtered off, washedwith THF (2×5 ml) and the combined filtrates were evaporated underreduced pressure. The solid residue was dissolved in ethyl acetate (250ml) and the solution was washed with 1M hydrochloric acid (3×80 ml),water (100 ml), 1 M sodium bicarbonate (3×80 ml) and brine (2×80 ml).The organic layer was dried over anhydrous magnesium sulphate andevaporated to dryness. The residue was dissolved in ethyl acetate andprecipitated with petroleum ether. Yield: 0.220 g (81%) of4-(benzyloxycarbonyl-L-leucylamino)-N-(tert-butoxycarbonyl)butylamine.

[α]²⁰ _(D)=−13.5° (c=1; MeOH), MS MALDI TOF: m/z 458.2 [M+Na]⁺, 474.1[M+K]⁺, calculated for C₂₃H₃₇N₃O₅: 435.6.

4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)-N-(tert-butoxycarbonyl)-butylamine

4-(benzyloxycarbonyl-L-leucylamino)-N-(tert-butoxycarbonyl)butylamine(0.210 g, 0,482 mmol) was dissolved in MeOH (25 ml). This solution washydrogenated over 10% Pd—C catalyst under atmospheric pressure during 1h at room temperature. Next, the catalyst was filtered off, washed withMeOH (10 ml) and the combined filtrates were evaporated. The resulted4-(L-leucylamino)-N-(tert-butoxycarbonyl)butylamine (0.199 g, 0.482mmol) was dissolved in 25 ml of THF and triethylamine (133 μl, 0.964mmol), next HOBt (0.065 g, 0.482 mmol) and Z-D-leucine (0.128 g, 0.482mmol) were added to the solution. The stirred mixture was cooled on icebath and DCC (0.109 g, 0.530 mmol) was added in small portions during0.5 h. The mixture was stirred in an ice bath for an additional 1 h,then left at room temperature overnight. The precipitated DCU wasfiltered off, washed with THF (2×5 ml) and the combined filtrates wereevaporated under reduced pressure. The solid residue was dissolved inethyl acetate (200 ml) and the solution was washed with 1M hydrochloricacid (3×70 ml), water (100 ml), 1 M sodium bicarbonate (3×70 ml) andbrine (2×70 ml). The organic layer was dried over anhydrous magnesiumsulphate and evaporated to dryness. The residue was dissolved in ethylacetate and precipitated with petroleum ether; yield: 0.219 g (83%) of4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)-N-(tert-butoxycarbonyl)butylamine.

[α]²⁰ _(D)=−1.0° (c=1; MeOH). MS MALDI TOF: m/z 571.4 [M+Na]⁺, 587.4[M+K]⁺, calculated for C₂₉H₄₅N₄O₆: 548.7.

N-amidino-4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)butylamine

4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)-N-(tert-butoxycarbonyl)-butylamine(0.209 g, 0.381 mmol) was dissolved in 8 ml of 4N solution of anhydroushydrochloride in dioxane. The reaction mixture was stirred during 0.5 hat room temperature, then evaporated to dryness under reduced pressure.The residual oil was triturated with anhydrous diethyl ether (25 ml).The obtained solid was filtered off under reduced pressure, washed withanhydrous diethyl ether (3×10 ml) and dried under vacuum over potassiumhydroxide. The resulted4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)butylamine hydrochloride(0.181 g, 0.374 mmol) was dissolved in 8 ml of DMF. Next, triethylamine(104 μl, 0.748 mmol) and 3,5-dimethyl-1-pyrazolylformamidinium nitrate(0.225 g. 1.12 mmol) were added. The pH of the mixture was kept about9.5. After five days the solution was evaporated and the resultedresidue was dissolved in 6 ml of 35% acetonitrile/water solutioncontaining 0.1% TFA (v/v/v). Half of the solution was injected on theRP-HPLC column (25×250 mm, Kromasil C-8, 5 μm) and eluted with 35%acetonitrile/water solution, containing 0.1% TFA (v/v/v) at flow rate 14ml/mm. (isocratic elution). The eluate was monitored using UV detectorat λ=226 nm. Fractions containing pureN-(amidino)-4-(benzyloxycarbonyl-L-leucyl-L-leucylamino)butylamine werecollected, concentrated under reduced pressure and lyophilized. Thesecond half of the crudeN-(amidino)-4-(benzyloxycarbonyl-L-leucyl-L-leucylamino)butylaminesolution was purified in the same manner.

Yield: 0.110 g (60%) of product as trifluoroacetate salt.

MS MALDI TOF: m/z 491.4 [M+H]⁺, 513.4 [M+Na]⁺, 529.4 [M+K]⁺, calculatedfor C₂₅H₄₂N₆O₄: 490.6.

N-amidino-4-(7-hydroxy-4-coumarinacetyl-D-leucyl-L-leucylamino)butylamine

N-Amidino-4-(benzyloxycarbonyl-D-leucyl-L-leucylamino)butylamine (0.100g, 0.2O4 mmol) was dissolved in MeOH (20 ml) and hydrogenated over 10%Pd—C catalyst under atmospheric pressure, during 1 h at roomtemperature. Next, the catalyst was filtered off and washed with MeOHThe combined filtrates were evaporated to dryness under reduced pressureto oil. Yield: 0,072 g (100%) ofN-(amidino)-4-(D-leucyl-L-leucylamino)butylamine.

To a solution of DIPEA (70 μl, 0.40 mmol), HOBt (0.027 g, 0.20 mmol),7-hydroxy-4-coumarinacetic acid (or (7-hydroxycoumarin-4-yl) aceticacid) (0.045 g, 0.20 mmol) in DMF (4 ml) andN-(amidino)-4-(D-leucyl-L-leucylamino)butylamine (0.072 g, 0.20 mmol),TBTU (0.065 g, 0.20 mmol) was added. After stirring during 12 h at roomtemperature, the solution was evaporated to dryness. The residue wasdissolved in mixture of solvents (1 ml DMF, 0.5 ml MeOH, 1.5 ml H₂O) andpurified by semipreparative reversed-phase HPLC on Kromasil column(25×250 mm, C-8, 5 μm), by isocratic elution with 15% acetonitrile/watersolution containing 0.1% TFA, at flow rate 14 ml/min. The eluate wasmonitored using UV detector at λ=226 nm. Fractions containing desiredproduct were concentrated under reduced pressure and lyophilized.

Yield: 11 mg (10%) ofN-(amidino)-4-(7-hydroxy-4-coumarinacetyl-D-leucyl-L-leucylamino)butylamineas trifluoracetate salt. The purity was confirmed by analytical HPLC(R_(t)=13.78 min in 30% acetonitrile/water solution containing 0.1% TFA)and MS IT-TOF analysis (m/z 559.8 [M+H]⁺; calculated for C₂₈H₄₂N₆O₆:558.7).

Example 2 Synthesis of Coumarinacetyl Derivative A-107(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino-2-(tert-butoxycarbonylamino)-3-methylbutane

The (2S)-1-amino-2-tert-butyloxycarbonylamino-3-methylbutanehydrochloride was obtained from tert-butyloxycarbonyl-L-valinol inaccordance with the literature procedures (Mattingly, P. G., Synthesis,1990, 1990, 366-368. Desired Boc-protected alcohol was obtained fromBoc-L-valine, in accordance with the literature procedure (Juszczyk, P.et al., Letters In Peptide Science. 2003, 10, 79-82). The solution of(2S)-1-amino-2-tert-butyloxycarbonylamino-3-methylbutane hydrochloride(0.50 g, 2.096 mmol), Z-L-phenylalanine (0.659 g, 2.20 mmol),triethylamine (611 μl, 4.4 mmol) and HOBt (0.297 g. 2.20 mmol) in 10 mlof DMF was cooled on ice bath and DCC (0.498 g, 2.42 mmol) was added insmall portions, during 0.5 h. The stirring was continued during 1 hour,next the reaction mixture was left in room temperature overnight. Theprecipitated DCU was filtered off, washed with DMF (2×3 ml) and thecombined filtrates were evaporated under reduced pressure. The solidresidue was dissolved in ethyl acetate (200 ml) and the solution waswashed with 1 M hydrochloric acid (3×70 ml),

water (70 ml), 1 M sodium bicarbonate (3×70 ml) and brine (3×70 ml). Theorganic layer was dried over anhydrous magnesium sulphate and evaporatedunder reduced pressure. The residue was dissolved in ethyl acetate andprecipitated with petroleum ether.

Yield: 0.743 g (85%) of(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(tert-butoxycarbonylamino)-3-methylbutane.

[α]²⁰ _(D)=−12.7° (c=1, methanol). MALDI-TOF analysis: m/z 506.2[M+Na]⁺, 522.2 [M+K]⁺ calculated for C₂₇H₃₇N₃O₅: 483.6.

(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(N-tert-butoxycarbonyl-L-leucylamino)-3-methylbutane

(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(tert-butoxycarbonylamino)-3-methylbutane(0.730 g, 1.51 mmol) was dissolved in 15 ml of 4N solution of anhydroushydrochloride in dioxane. The reaction mixture was stirred during 0.5 hat room temperature, then evaporated under reduced pressure. Theresidual oil was triturated with anhydrous diethyl ether (50 ml). Theobtained solid was filtered off under reduced pressure, washed withanhydrous diethyl ether (3×10 ml) and dried under vacuum over potassiumhydroxide. The resulted(2S)-2-amino-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(tert-butoxycarbonyl-amino)-3-methylbutanehydrochloride (0.622 g, 1.48 mmol) was dissolved in 14 ml DMF/DCM (5:9v/v) mixture, then triethylamine (412 μl, 2.96 mmol), HOBt (0.200 g,1.48 mmol) and Boc-L-leucine (0.342 g, 1.48 mmol) were added to thesolution. The mixture was cooled on ice bath and DCC (0.336 g, 1.63mmol) was added in small portions during 0.5 h, with vigorous stirring.Stirring on ice bath was continued during additional 1 h, then thereaction mixture was left at room temperature overnight. Theprecipitated DCU was filtered off, washed with DMF (5 ml) and thecombined filtrates were evaporated under reduced pressure. The solidresidue was dissolved in 250 ml of ethyl acetate and the solution waswashed with 1 M hydrochloric acid (3×80 ml), water (100 ml), 1 M sodiumbicarbonate (3×80 ml) and saline (3×80 ml). The organic layer was driedover anhydrous magnesium sulphate and evaporated to dryness. The residuewas dissolved in hot toluene and precipitated with petroleum ether,yielding 0.795 g (90%) of(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(N-tert-butoxycarbonyl-L-leucylamino)-3-methylbutane.

[α]²⁰ _(D)=−21.6° (c=1, methanol). MALDI-TOF analysis: m/z 619.2[M+Na]⁺,635.2 [M+K]⁺ calculated for C₃₃H₄₈N₄O₆: 596.8.

(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-3-methylbutane

(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-[(N-tert-butoxycarbonyl)-5-aminopentanoyl-L-leucylamino]-3-methylbutane(0.785 g, 1.13 mmol) was dissolved in 15 ml of 4N solution of anhydroushydrochloride in dioxane. The reaction mixture was stirred during 0.5 hat room temperature, then evaporated to dryness under reduced pressure.The residual oil was triturated with anhydrous diethyl ether (50 ml).Obtained solid was filtered off under reduced pressure, washed withanhydrous diethyl ether (3×10 ml) and dried under vacuum, over potassiumhydroxide. The resulted(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-(L-leucylamino)-3-methylbutanehydrochloride (0.687 g, 1.29 mmol) was dissolved in 10 ml DMF/DCM (v/v5:9) and triethylamine (359 μl, 2.58 mmol), HOBt (0.174 g, 1.29 mmol),N-tert-butoxycarbonyl-5-aminopentanoic acid (0.275 g, 1.29 mmol) wereadded to the solution. The mixture was cooled in an ice bath, then DCC(0.292 g, 1.42 mmol) was added in small portions, during 0.5 h withvigorous stirring. The mixture was stirred in an ice bath for additional1 h, then left at room temperature overnight. The precipitated DCU wasfiltered off, washed with DMF (5 ml) and the combined filtrates wereevaporated under reduced pressure. The solid residue was dissolved in250 ml of ethyl acetate and the solution was washed with 1 Mhydrochloric acid (3×80 ml), water (100 ml), 1 M sodium bicarbonate(3×80 ml) and saline (3×80 ml). The organic layer was dried overanhydrous magnesium sulphate and evaporated to dryness. The residue wasdissolved in hot toluene and precipitated with petroleum ether, yielding0.817 g (96%) of(2S)-1-(N-benzyloxycarbonyl-L-phenylalanylamino)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-3-methylbutane.

[α]_(D) ²⁰ =−17.2° (c=1, methanol). MALDI-TOF analysis: m/z 718.0[M+Na]⁺, 733.9 [M+K]⁺; calculated for C₃₈H₅₇N₅O₇: 695.9.

(2S)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-1-[N-(7-hydroxy-4-coumarinacetyl)-L-phenylalanylamino]-3-methylbutane

(2S)-1-(N-butoxycarbonyl-L-phenylalanylamino)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-3-methylbutane(0.800 g. 1.15 mmol) was dissolved in methanol (25 ml) and hydrogenatedover 10% Pd—C catalyst under atmospheric pressure during 1 h, at roomtemperature. Next, the catalyst was filtered off and washed with MeOHand the combined filtrates were evaporated to dryness under reducedpressure to oil. Yield: 0.646 g (100%).

To cooled on ice bath and vigorously stirred solution of(2S)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-1-(L-phenylalanylamino)-3-methylbutane(0.646 g. 1.15 mmol), 7-hydroxy-4-coumarinacetic acid (0.253 g, 1.15mmol), HOBt (0.155 g, 1.15 mmol), DIPEA (394 μl, 2.30 mmol) in DMF (10ml), DCC (0.261 g, 1.27 mmol) was added in small portions, during 0.5 h.The mixture was stirred on ice bath for additional 1 h, next left atroom temperature overnight. The precipitated DCU filtered off, washedwith DMF (5 ml) and the combined filtrates were evaporated under reducedpressure. Obtained solid residue was dissolved in ethyl acetate (250 ml)and the solution was washed with 1 M hydrochloric acid (3×80 ml), water(100 ml), 1 M sodium bicarbonate (3×80 ml) and brine (3×80 ml). Theorganic layer was dried over anhydrous magnesium sulphate and evaporatedto dryness. The residue was dissolved in hot toluene and precipitatedwith petroleum ether, Yield: 0.799 g (91%) of(2S)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-1-[N-(7-hydroxy-4-coumarinacetyl)-L-phenylalanylamino]-3-methylbutane;

[α]_(D) ²⁰ =−16.0° (c=1, methanol). MS IT-TOF analysis: m/z 764.4[M+H]⁺; calculated for C₄₁H₅₇N₅O₉: 763.4.

(2S)-2-[N-(5-guanidinepentanoyl)-L-leucylamino]-1-[N-(7-hydroxy-4-coumarinylacetyl-L-phenylalanylamino)-3-methylbutane

(2S)-2-[N-tert-butoxycarbonyl-5-aminopentanoyl-L-leucylamino]-1-[N-(7-hydroxy-4-coumarinacetyl-L-phenylalanylamino)-3-methylbutane(0.100 g, 0.131 mmol) was dissolved in 10 ml of 4N solution of anhydroushydrochloride in dioxane. The reaction mixture was stirred during 0.5 hat room temperature and then evaporated to dryness under reducedpressure. The residual oil was triturated with anhydrous diethyl ether(30 ml). The obtained solid was filtered off under reduced pressure,washed with anhydrous diethyl ether (2×10 ml) and dried under vacuumover potassium hydroxide. The resulted(2S)-2-(N-(5-aminopentanoyl)-L-leucylamino)-1-[N-(7-hydroxy-4-coumarinylacetyl)-L-phenylalanylamino]-3-methylbutane(0.099 g, 0.142 mmol) was dissolved in 5 ml of DMF/MeOH (v/v, 1:1).Next, triethylamine (39 μl, 0.28 mmol),3,5-dimethyl-1-pyrazolylformamidinium nitrate (0.086 g, 0.43 mmol) wasadded. The pH of the mixture was kept about 9.5. After five days, thesolution was evaporated and the resulted residue was dissolved in 3 mlof mixture DMSO/water (2:1, v/v). The solution was injected on theRP-HPLC column (25×250 mm, Kromasil C-8, 5 μm) and eluted with 30%acetonitrile/water solution containing 0.1% TFA (isocratic elution), atflow rate 14 ml/mm. The eluate was monitored using UV detector at λ=226nm. Fractions containing pure desired product were collected,concentrated under reduced pressure and lyophilized.

Yield: 9 mg (9%) of(2S)-2-[N-(5-guanidinepentanoyl)-L-leucylamino]-1-[N-(7-hydroxy-4-coumarinylacetyl-L-phenylalanylamino]-3-methylbutaneas trifluoroacetate salt.

The purity was confirmed by analytical HPLC(R_(t)=15.11 min. in 36%acetonitrile/water/0.1% TFA solution) and MS IT-TOF analysis (m/z 706.2[M+H]⁺; calculated for C₃₇H₅₁N₇O₇: 705.4).

Example 3

Inhibition of Osteoclast Formation

Spleen Cell Cultures

Cells were obtained from spleens of 5- to 9-week-old mice. The spleenswere dissected free of adhering tissues, and cells were released byrubbing the spleens against the bottom of a Petri dish, in which grooveshad been made by a scalpel. Erythrocytes were lysed in red blood celllysis buffer (0.16 MNH4Cl, 0.17 M Tris, pH7.65) and the remaining cellswere seeded, at a cell density of 106 cells/cm2, on plastic coverslipsplaced in 24-well plates. The cells were cultured in 0.5 ml a-minimalessential medium (a-MEM) supplemented with 10% fetal bovine serum (FBS),L-glutamine (0.7 mM), 100 U/ml benzylpenicillin, 100 mg/ml streptomycinand 100 mg/ml gentamycin sulphate. Cells were allowed to settleovernight in complete medium and thereafter the medium was changed andthe experiment started. Osteoclast precursor cells were induced toproliferate and differentiate by the addition of M-CSF (25 ng/ml) andRANKL (100 ng/ml). To study the effect of the compounds onosteoclastogenesis, cells were grown in medium containing compounds atdifferent concentrations, together with M-CSF and RANKL. Cells culturedin complete medium without M-CSF and RANKL were included in allexperiments as a control. Medium was changed after 3 days. After 1-6days, the cells were washed three times with PBS (pH 7.35) and fixedwith acetone in citrate buffer/3% formaldehyde solution. Cells were thenstained for tartrate-resistant acid phosphatase (TRAP) activity usingthe SigmaDiagnostics Acid Phosphatase Leukocyte staining kit and byfollowing the manufacturer's instruction. Multinucleated (no. ofnuclei>3), TRAP-positive cells were counted as osteoclasts(TRAPC-MuOCL). Osteoclasts formed in the spleen cell cultures stimulatedby M-CSF and RANKL were able to form pits when cultured on slices ofdevitalised bovine bone and the stimulation of osteoclastogenesis wasassociated with increased mRNA expression of several osteoclast genesincluding ctr, trap and cathepsin K. No osteoclasts were formed whencells were treated with either M-CSF or RANKL alone and the stimulationcaused by M-CSF and RANKL was abolished by osteoprotegerin (OPG). Noosteoclasts were formed in the presence of PTH or 1,25(OH)2-vitamin D3(D3) indicating the lack of stromal cells in the spleen cell cultures.

Bone Marrow Macrophage Cultures

Highly purified BMMs were isolated according to Takeshita et al. (2000).These cells did not express alkaline phosphatase, RANKL, OPG or CTRmRNA, but mRNA for RANK, c-Fms, cathepsin K and TRAP, as assessed byquantitative real-time PCR (data not shown).

For osteoclastogenesis experiments, BMMs were seeded either on 0.8 cm²glass chamber slides or 0.32 cm² 96-well plates at a density of 10⁴cells/cm² in a-MEM/10% FBS containing either 100 ng/ml M-CSF (controls)or 100 ng/ml M-CSF C50 ng/ml RANKL, with and without test compounds.After 4-5 days, with a change of medium after 3 days, the cultures wereharvested and the cells fixed with acetone in citrate buffer/3%formaldehyde and subsequently stained for TRAP. The TRAP-positive cellswith three or more nuclei were considered osteoclasts, and the number ofmultinucleated osteoclasts was counted (TRAP⁺-MuOCL). Osteoclasts formedin these cultures stimulated by M-CSF and RANKL were able to form pitswhen cultured on slices of bovine bone and osteoclast formation wasassociated with increased mRNA expression of CTR, TRAP and cathepsin K.Osteoclasts were not formed in the presence of PTH or D3, indicating thelack of stromal cells in the cultures. Osteoclast formation caused byM-CSF and RANKL was abolished by OPG

TABLE 1 BMM #27 + BMM #29 + BMM #30 + BMM #34. Four differentexperiments with purified bone marrow macrophages stimulated with M- SCFand RANKL to induce formation of trap+ multinucleated osteoclasts withand without different compounds. Papain Cath K Cath B Osteoclastformation Comp. Ki nM Ki nM Ki nM (100% = M-CSF + RANKL) A-25 Inactive42.2 8.1 1% A-11 93 14 360 1% A-12 94 13 1000 2% A-33 46 24 2 0% A-16340 inactive inactive 16%  A-47 16 99 0.3 0% A-42 161 inactive 14 0%A-50 68 63 55 2% A-54 1000 14 228 1% Z-RLR 9 1 0.0005 0% A-20 InactiveNd Nd 0% M-1 23 160 3 14%  A-40 Nd Nd Nd 0% A-49 Nd Nd Nd 0.2%   A-55 NdNd Nd 0.2%   A-59 Nd Nd Nd 0%

TABLE 2 BMM #141 and BMM #142. experiments with purified bone marrowmacrophages stimulated with M-SCF and RANKL to induce formation of trap+multinucleated osteoclasts with and without different compoundsOsteoclast formation Comp. (100% = M-CSF + RANKL) A-107 0% A-127 16%A-128 4% A-52 15% A-55 8% Cystatin C 0%

The invention claimed is:
 1. A method of treating a condition associatedwith bone loss or low bone density or for inhibiting osteoclastdifferentiation and stimulation or bone resorption in a mammal in needthereof, comprising administering to said mammal an effective amount ofone or more compounds selected from the group consisting of A-20, A-33,A-42, A-47, A-59, and A-107:

the one or more compound being a single stereoisomer or a mixture ofdifferent stereoisomers, or a pharmaceutically acceptable salt thereof.2. The method according to claim 1, wherein the compound is administeredas a pharmaceutical composition comprising a pharmaceutically acceptablecarrier, buffer, excipient or diluent.
 3. The method according to claim1, wherein the one or more compound is administered in a prostheticdevice.
 4. The method according to claim 1, wherein the condition is abone disease selected from the group consisting of osteoporosis,juvenile osteoporosis, osteogenesis imperfecta, hypercalcaemia,hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bonedisease, osteonecrosis and Paget's disease of bone, or the condition isa secondary osteoporosis of bone loss due to rheumatoid arthritis,inflammatory arthritis, osteomyelitis, bone loss due to an eatingdisorder, metastatic bone diseases, periodontal bone loss, bone loss dueto cancer, age-related loss of bone, or bone resorption induced bypharmaceuticals or implants.
 5. A method of treating a subject sufferingfrom a high level of bone resorption, comprising administering to thesubject an effective amount of one or more compounds selected from thegroup consisting of A-20, A-33, A-42, A-47, A-59, and A-107, as definedin claim
 1. 6. A method of inhibiting osteoclast formation or activityin a subject in need thereof, comprising administering to the subject aneffective amount of one or more compounds selected from the groupconsisting of A-20, A-33, A-42, A-47, A-59, and A-107, as defined inclaim
 1. 7. The method according to claim 6, wherein the subject issuffering from osteoporosis.
 8. The method according to claim 1, whereinthe one or more compounds are selected from the group consisting ofA-33, A-42, A-47, A-59, and A-107.
 9. The method according to claim 1,wherein the method is for treating a condition associated with bone lossor low bone density, and the one or more compounds are selected from thegroup consisting of A-33, A-42, A-47, A-59, and A-107.